Flaviviruses are (+) RNA viruses that cause such diseases as West Nile fever (“WN”), dengue fever (“DEN”), yellow fever (“YF”), St. Louis encephalitis, Japanese encephalitis (“JE”), and tick-borne encephalitis (“TBE”). West Nile virus was first isolated over 60 years ago from the blood of a febrile patient (Smithburn et al., 1940), and is one of the most widespread flaviviruses worldwide. The virions of the West Nile fever virus are spherical particles with a diameter of 50 nm constituted by a lipoproteic envelope surrounding an icosahedric nucleocapsid containing a positive polarity, single-strand RNA. A single open reading frame (“ORF”) encodes all the viral proteins in the form of a polyprotein. The cleaving and maturation of this polyprotein leads to the production of about ten different viral proteins. The structural proteins are encoded by the 5′ part of the genome and correspond to the nucleocapsid designated C (14 kDa), the envelope glycoprotein designated E (50 kDa), the pre-membrane protein designated prM (23 kDa), the membrane protein designated M (7 kDa). The non-structural proteins are encoded by the 3′ part of the genome and correspond to the proteins NS1 (40 kDa), NS2A (19 kDa), NS2B (14 kDa), NS3 (74 kDa), NS4A (15 kDa), NS4B (29 kDa), NS5 (97 kDa).
The West Nile virus is endemic to Africa and has been repeatedly registered in Europe and Asia for decades causing self-limiting epidemics and epizootics (Murgue et al., 2001; Savage et al., 1999). Recent introduction of the virus into the naïve environment of the North American continent (Lanciotti et al., 1999) had disastrous consequences both for wildlife and human population (Roehrig et al., 2002) and in a few years has developed into a nationwide epidemiological problem. In addition to hundreds of human mortality cases reported to the Centers for Disease Control (CDC, 2004), the virus imposes a substantial economical burden, especially on the equine industry (Anonymous, 2003).
Cell-mediated immune response plays an important role in virus clearance and in protection from the disease (Diamond et al., 2003; Shrestha and Diamond, 2004). Since flavivirus nonstructural proteins supply the majority of dominant T-cell peptide determinants (Co et al., 2002), cell-mediated response induced by chimeric flaviviruses are mostly to vector proteins. However, development of a live attenuated West Nile vaccine, which may have a better capability to elicit balanced humoral and cell-mediated immune responses, is hindered by the high virulence and pathogenicity of the NY99 strain circulating in the U.S. (Beasley et al., 2002; Roehrig et al., 2002).
Based on serological data and genetic characterization, West Nile viruses were subdivided into two distinct lineages (Berthet et al., 1997; Price and O'Leary, 1967). Viruses of lineage 1, which includes the highly virulent NY99 strain, are most widespread and often were found in association with epidemics or epizootics (Roehrig et al., 2002). Although a few strains with a high virulence were also found among lineage 2 representatives (Beasley et al., 2002), viruses of this lineage have not been associated with disease outbreaks (Lanciotti et al., 1999). For this reason, lineage 2 viruses may be more attractive for development of live attenuated West Nile vaccine.
Modern (+) RNA virus studies increasingly rely on the infectious clone methodology, which allows for targeted manipulation of viral genomes. In the “classical approach”, (+) RNA viruses are recovered from cells transfected with synthetic RNA made by in vitro transcription of infectious clone cDNA templates. In a layered DNA/RNA approach, also known as “infectious DNA,” the infectious (+) RNA viruses are recovered directly after transfection of plasmids carrying viral genome cDNA into susceptible cells. Unfortunately, difficulties are often encountered in the design of flavivirus infectious DNA. Few studies have reported on the use of such infectious DNA construct as a vaccine.
The present invention is directed to the isolation of a virus useful for development of a West Nile vaccine. Isolate 956D117B3 (earlier also referred to as WN-Nigeria or WN-Wengler (Berthet et al., 1997; Lanciotti et al., 1999)) is a descendant of the West Nile virus prototype B956 (Smithburn et al., 1940), and is one of the first flaviviruses for which the complete nucleotide sequence has been determined (Castle et al., 1986; Castle et al., 1985; Wengler et al., 1985; GenBank #M12294). Earlier, the first West Nile infectious clone designed on the basis of the isolate 956D117B3 (Yamshchikov et al., 2001) was reported. While the virus was produced, the viral population was not characterized. In the present invention, it was demonstrated for the first time that the virus recovered from the molecular clone is highly attenuated, induces vigorous and balanced immune response and even at low doses protects mice against the virulent NY99 strain. Combined with its stable genotype and excellent growth characteristics in tissue culture, the recovered virus is well-suited for the development of veterinary and human live West Nile vaccines.